We investigated the site-specific local structure of an amyloid peptide, NH(2)-GSNKGAIIGLM-COOH [Aβ(25-35)], one of the active fragments of amyloid β peptide that is known to be responsible for Alzheimer's disease, in the fibrillar aggregated state. Isotope-assisted infrared vibrational circular dichroism (VCD) and absorption (VA) spectroscopy were used for the parent Aβ(25-35) peptide, along with doubly (13)C labeled peptides at the carbonyl groups of residues 29 (Gly) and 30 (Ala) [Aβ(25-35:(13)C-29/30)] and at the carbonyl groups of residues 33 (Gly) and 34 (Leu) [Aβ(25-35:(13)C-33/34)]. The present results confirm that Aβ(25-35) peptide fibrils adopt a β-sheet structure and isotopic dilution experiments suggest a parallel β-sheet structure. The isotopic shifts suggest that the microenvironment of residues 29 (Gly) and 30 (Ala) could be different from that of residues 33 (Gly) and 34 (Leu). An unusual enhancement for the amide II' VCD intensities of Aβ(25-35:(13)C-29/30) and Aβ(25-35:(13)C-33/34) peptide fibrils, considered to originate from inter-strand coupling, was found for the first time. The structural information reported in this manuscript has important implications in understanding the role of this peptide in the development of Alzheimer's disease.